Technical Information

Protocol for the Preparation and Fluorescent ICC Staining of Non-adherent Cells

The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for fluorescent ICC experiments using cell smears.

This protocol provides a basic guide for the preparation, fixation, and fluorescent staining of cell smear samples. Each investigator must determine the precise experimental conditions required to generate a strong and specific signal for each antigen of interest. If R&D Systems primary antibodies are employed, please refer to the product data sheets to obtain the recommended working dilutions. In the staining protocol, signal visualization is achieved using R&D Systems NorthernLights™ range of fluorescent secondary antibodies and reagents. For all other reagents, please follow the manufacturer’s instructions.

Please read the protocol in its entirety before starting.

Protocol for the Preparation of a Cell Smear for Non-adherent Cell ICC

Smearing non-adherent cells across a gelatin-coated slide forms a monolayer of cells that can be easily visualized by ICC.

Dilute the unconjugated primary antibody (or fluorescence-conjugated primary) in dilution buffer according to the manufacturer’s instructions. For fluorescent ICC staining of cell smears using R&D Systems antibodies, it is recommended to incubate at room temperature for 1 hour. Alternatively, incubate overnight at 2-8 °C.Note: Appropriate controls are critical for the accurate interpretation of IHC/ICC results. All IHC/ICC experiments should include a negative control using the incubation buffer with no primary antibody to identify non-specific staining of the secondary reagents. Additional controls can be employed to support the specificity of staining generated by the primary antibody. These include absorption controls, isotype matched controls (for monoclonal primary antibodies), and tissue type controls.

Wash two times in 400 µL of wash buffer. If using a primary antibody with a direct fluorescent conjugate, go to step 8.

Dilute the secondary antibody in dilution buffer according to the manufacturer’s instructions. Add 400 µL to the wells, and incubate at room temperature for 1 hour in the dark. From this step forward, samples should be protected from light.

Note: R&D Systems NorthernLights fluorescent secondary antibodies and streptavidin conjugates are bright, resistant to photobleaching, and are ideal for multi-color fluorescence microscopy.Note: If a biotinylated antibody was used in step 4, apply the appropriate NorthernLights Streptavidin conjugate in step 6.

Rinse two times in 400 µL of wash buffer.

Add 300 µL of the diluted DAPI solution to each well, and incubate 2-5 minutes at room temperature. DAPI binds to DNA and is a convenient nuclear counterstain. It has an absorption maximum at 358 nm and fluoresces blue at an emission maximum of 461 nm.Note: DAPI counterstain can obscure visualization of targets localized in cell nuclei.

Rinse once with PBS and once with water.

Carefully remove the coverslips from the wells and blot to remove any excess water. Dispense 1 drop of anti-fade mounting medium onto the microscope slide per coverslip.

Visualize using a fluorescence microscope and filter sets appropriate for the label used. Slides can also be stored in a slide box at < -20 °C for later examination.Note: Initial IHC/ICC studies often require further optimization and/or additional troubleshooting steps.